Studies on the functional modulating effect of redox partners on the cytochrome P450 enzyme MycG

YANG Chaofan; JIANG Yuchao; SANG Moli; LI Shengying; ZHANG Wei

doi:10.12211/2096-8280.2021-053

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Studies on the functional modulating effect of redox partners on the cytochrome P450 enzyme MycG

Research Article | 更新时间：2025-03-20

- Studies on the functional modulating effect of redox partners on the cytochrome P450 enzyme MycG
- Synthetic Biology Journal Vol. 3, Issue 3, Pages: 587-601(2022)
- 作者机构：
  
  山东大学微生物技术研究院，山东大学微生物技术国家重点实验室，山东青岛 266237
- 作者简介：
- 基金信息：
- DOI：10.12211/2096-8280.2021-053
  CLC： Q936
- Received：30 April 2021，
  
  Revised：2021-06-08，
  
  Published：30 June 2022
- 稿件说明：
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杨超凡，姜玉超，桑茉莉，李盛英，张伟. 还原伴侣对细胞色素P450酶MycG功能调控的研究［J］. 合成生物学， 2022， 3（3）： 587-601

YANG Chaofan， JIANG Yuchao， SANG Moli， LI Shengying， ZHANG Wei. Studies on the functional modulating effect of redox partners on the cytochrome P450 enzyme MycG［J］. Synthetic Biology Journal， 2022， 3（3）： 587-601
杨超凡，姜玉超，桑茉莉，李盛英，张伟. 还原伴侣对细胞色素P450酶MycG功能调控的研究［J］. 合成生物学， 2022， 3（3）： 587-601 DOI： 10.12211/2096-8280.2021-053.

YANG Chaofan， JIANG Yuchao， SANG Moli， LI Shengying， ZHANG Wei. Studies on the functional modulating effect of redox partners on the cytochrome P450 enzyme MycG［J］. Synthetic Biology Journal， 2022， 3（3）： 587-601 DOI： 10.12211/2096-8280.2021-053.

摘要

细胞色素P450酶负责催化许多天然产物生物合成过程中的关键反应，是自然界中最具催化多样性的生物催化剂。还原伴侣蛋白对P450催化功能至关重要，在进化过程中两者之间通过融合或分离方式演化出多种不同的催化系统。还原伴侣与P450酶的适配性往往是造成P450酶功能重建失败或低效的关键因素，也能够影响和改变P450酶的催化功能和性质。不同还原伴侣与P450酶的组合与互作如何影响P450酶的催化功能、催化效率和产物分布是值得深入探究的科学问题。本研究以还原伴侣蛋白RhFRED和P450酶MycG为研究对象，通过构建不同的融合和分离蛋白组合，系统研究了还原伴侣与P450酶的适配和相互作用模式对P450酶催化功能的影响。将RhFRED两个结构功能域拆解为两个独立蛋白FMN和Fe

，与P450酶MycG三者之间利用还原伴侣工程构建分离或融合型蛋白，通过体外生化反应探究不同组合方式下MycG对底物麦新米星M-Ⅳ催化功能以及电子传递效率的影响。在构建的20个还原伴侣与P450酶的“非天然”催化组合中，16个组合能够成功重建MycG的催化功能，其中12个组合中MycG能够催化产生3种氧化产物麦新米星M-Ⅰ、M-Ⅱ和M-Ⅴ以及1种脱甲基产物dMe-M-Ⅳ；4个组合仅产生氧化产物M-Ⅰ、M-Ⅱ和M-Ⅴ；剩余4个组合未检测到任何反应产物。通过模拟自然进化，系统研究了还原伴侣与P450酶构成的3种不同催化系统（单、双、三组分）以及单组分和双组分系统中不同的蛋白组织形式对MycG催化功能和性质的影响，结果表明不同还原伴侣系统和组合/组织方式能够通过蛋白-蛋白相互作用显著影响P450的催化功能、催化效率和催化性质。

Abstract

Cytochrome P450 enzymes

which are widely involved in the key biosynthetic steps of many natural products

are considered as the most versatile biocatalysts in the nature. Redox partners responsible for electron transfer are indispensable for most P450 catalytic reactions. During the long evolution process

various P450-redox partner systems have been constituted by protein fusion and recombination. The protein-protein interaction and recognition between P450 and redox partner(s) are not only the key factor for functional reconstitution of P450s

but also can affect and change the catalytic functions and properties of P450 enzymes. To address the issue that how the choice and recombination of different redox partners affect the catalytic behaviors (reaction type

catalytic efficiency

product distribution

and electron transport) of the P450 enzyme

a number of fusion

and separation P450 systems were constructed based on the two-domain redox partner protein RhFRED and P450 MycG. Specifically

the two domains of RhFRED were separated and expressed as stand-alone FMN and Fe

proteins. The positions of these two domains and MycG were shuffled and engineered.

In vitro

biochemical reactions were carried out to explore the effect of different redox partner combinations on the MycG-catalyzed bioconversion of mycinamicin-Ⅳ (M-Ⅳ) and the electron transfer efficiency as well. As results

among the 20 tested catalytic systems

the functions of the multifunctional P450 enzyme MycG were successfully reconstituted through 12 combinations

in which three oxidative products including M-Ⅰ

M-Ⅱ and M-Ⅴ

and one demethylation product dMe-M-Ⅳ were produced. Besides

4 combinations only led to the production of three oxidative products. In addition

no products were detected in the other 4 combinations. By simulating natural evolutionary strategy

the effects of three different catalytic systems of redox partner(s) and P450 enzymes

together with different protein organization forms on the functional modulating of MycG

were studied systematically. These results indicate that the P450 catalytic properties are affected and modulated by not only the identity

but also the position and organization of redox partner (s)

probably

via

alternative protein-protein interactions.

关键词

Keywords

references

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杨超凡

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山东大学微生物技术研究院，山东大学微生物技术国家重点实验室

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